The study of retinoblastoma, a childhood tumor, has provided numerous insights into cancer biology. Perhaps most significantly, this work has revealed that cancers can develop in response to mutations in tumor suppressor genes. Tumor suppressor mutations account for ~80% of all cancer mutations, but unlike activating mutations in oncogenes, cannot be targeted by drugs that inhibit a mutant gene product. The Cobrinik lab has recently shown that retinoblastoma growth is driven not only by retinoblastoma protein (RB) mutations, but also by normal signaling circuitry of retinal cone precursors. This normal circuitry includes extremely high expression of murine double minute oncogene (MDM2), V-myc myelocytomatosis viral related oncogene (MYCN), thyroid hormone receptor 2 (THRB2) and retinoic acid receptor gamma (RXRG) nuclear receptor. The expression of these genes is critical to retinoblastoma cell growth and survival, suggesting that they may be effective therapeutic targets despite the fact that they are not genetically altered in retinoblastoma. MYCN is an especially attractive target for development because it drives tumorigenesis in diverse cancers, including neuroblastoma, medulloblastoma, and other CNS malignancies. Moreover, transient MYC down-regulation appears to be well tolerated at the organismal level, while dramatically suppressing tumorigenesis. However, MYCN proteins are considered to be 'undruggable' at the present time. siRNA-based drugs represent a new and potentially significant therapeutic paradigm for the treatment of multiple diseases including cancer and retinal disorders. The ability to apply this technology to human disease has been impeded by the absence of efficient and non-toxic in vivo delivery systems. We have developed a novel class of hydrophobically modified RNAi compounds, self-delivering rxRNA or sd-rxRNATM, that do not require a delivery vehicle to enter cells and have improved pharmacology compared to traditional siRNAs. Intravitreal administration of sd-rxRNA in a rodent eye results in complete retina penetration and efficient tumor uptake. In addition, potent long lasting silencing (up to 21 days post single injection) was demonstrated. The objective of this proposal is to develop novel sd-rxRNA based therapeutics for treatment of retinoblastoma and other cancers. We believe that sd-rxRNA based treatment of retinoblastoma is feasible and will have minimal systemic toxicity which is specifically important in the context of pediatric patients. As part of this proposal, sd-rxRNA distribution via different routes of administration (intravitreal and intra-arterial injection) and target mRNA silencing in retinoblastoma cell tumors seeded into the eye will be evaluated in vivo. In parallel, sd-rxRNA compounds against MYCN and other retinoblastoma targets (as back up) will be identified and confirmed to be efficacious in vitro. A phase II application will focus on silencing of MYCN and these other targets in a retinoblastoma model. The impact of silencing on progression of a human tumor xenograft in mouse eye will be evaluated. The most potent compounds will be moved into preclinical development with a focus on developing a data package sufficient to support filing an IND. PUBLIC HEALTH RELEVANCE: Retinoblastoma is cancer of the eye that usually begins in the retina and primarily affects young children. Current treatments include chemotherapy, radiation therapy, laser therapy and possible surgery. We have recently developed a novel class of hydrophobically modified RNAi compounds and have demonstrated potent and long lasting silencing in a rodent eye. The focus of this grant application is to evaluate the applicability of d-rxRNA to silence genes in a retinoblastoma model in vivo.